Research ArticleCell Biology

Phosphorylation of the exocyst protein Exo84 by TBK1 promotes insulin-stimulated GLUT4 trafficking

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Science Signaling  21 Mar 2017:
Vol. 10, Issue 471, eaah5085
DOI: 10.1126/scisignal.aah5085
  • Fig. 1 TBK1 is required for insulin-stimulated GLUT4 trafficking.

    (A) 2-DG uptake was assessed in cells transfected with the indicated siRNAs. Data are means ± SEM. *P < 0.05; n = 3 experiments. SC, scrambled control. (B) Immunoblots (IBs) from (A) show siRNA-mediated knockdown efficiencies of IKKα, IKKβ, IKKγ, and TBK1 in 3T3-L1 adipocytes treated with or without 10 nM insulin for 30 min. n = 3 experiments. p, phosphorylated. (C) Immunostaining of 3T3-L1 adipocytes stably expressing MYC-GLUT4-eGFP electroporated with siRNA against TBK1 and stimulated with 10 nM insulin for 15 min. MYC (red) in nonpermeabilized cells indicates GLUT4 translocation to the plasma membrane. GFP fluorescence in cells indicate total GLUT4 expressed. A merge of GFP and MYC is shown. Scale bar, 200 μm. n = 3 experiments. (D) Percentage of cells that undergo GLUT4 translocation as shown in (C). Number of cells that show MYC staining at the rim as a percentage of GFP-positive cells was counted for scrambled and TBK1 knockdown. Data are means ± SEM. *P < 0.05; n = 97 to 105 cells per group. (E) Immunoblot showing knockdown efficiencies of TBK1 from (D). (F) 3T3-L1 adipocytes pretreated with inhibitors of Akt (Akti) and TBK1 (amlexanox) before treatment with insulin (Ins) were subjected to 2-DG uptake assy. Data are means ± SEM. *P < 0.05; n = 3 experiments; data were analyzed by Mann-Whitney U test. (G) Immunoblot from (C). n = 3 experiments. DE, dark exposure; LE, light exposure.

  • Fig. 2 Exo84 is a TBK1 substrate in vitro and in cells.

    (A) GST-Exo84 full-length wild-type (WT) or RBD was incubated with recombinant His-TBK1 WT and [γ-32P]adenosine triphosphate (ATP). Immunoblots were stained with Ponceau S (top panel) or subjected to autoradiography (bottom panel). Immunoblot shows the amount of His-TBK1 WT used for the assay. n = 3 experiments. (B) GST-free Exo84 RBD (lanes 1 to 6), GST-Exo84 WT (lane 7), GST (lane 8), or none (lane 9) was incubated with MBP-TBK1 in the presence of [γ-32P]ATP. MBP-TBK1 (0.1 to 4.0 μg) was used in lanes 1 to 6, and 0.5 μg of MBP-TBK1 was used in lanes 7 to 9. Phosphorylation of proteins was detected by autoradiography. n = 3 experiments. (C) Various kinases were incubated with MBP (top panel) or GST-Exo84 WT (bottom panel) in the presence of [γ-32P]ATP. Phosphorylation of proteins was detected by autoradiography. Asterisk indicates phosphorylated GST-Exo84 WT. Immunoblot shows the amount of GST-Exo84 WT used for the assay. n = 3 experiments. C, catalytic subunit. (D) Immunoblot showing reduced electrophoretic mobility of Exo84 when incubated with TBK1 WT but not the kinase-inactive K38A mutant. n = 3 experiments. (E) MYC-Exo84 was immunoprecipitated (IP) from COS-1 cells coexpressing TBK1 and IKKε or the kinase-inactive mutants. Anti-MYC immunoprecipitates were then treated with or without CIP. n = 3 experiments.

  • Fig. 3 TBK1 directly interacts with Exo84 through the coiled-coil domain of TBK1 and helical domain (Helical D) of Exo84.

    (A) Schematic representation of various domains in full-length and truncated Exo84. (B) Coimmunoprecipitates of hemagglutinin (HA)–tagged various truncation mutants of Exo84 with Flag-TBK1 WT or the K38A kinase-inactive mutant from COS-1 cells. n = 3 experiments. WCL, whole-cell lysate.

  • Fig. 4 Phosphorylation of Exo84 by TBK1 decreases its interaction with RalA.

    (A) Coimmunoprecipitates of Flag-RalA WT or G23V mutant with HA-Exo84 WT in the presence or absence of TBK1 WT or the K38A kinase-inactive mutant from COS-1 cells. Arrow indicates immunoprecipitated Exo84. Quantification of the binding of Exo84 with RalA G23V is shown. Binding was normalized to the fourth lane. The normalized ratio is represented in arbitrary units (AU). Data are means ± SEM. *P < 0.05; n = 3 experiments. (B) Cell lysates from COS-1 cells coexpressing increasing amounts of Flag-tagged WT TBK1 and the K38A kinase-inactive mutant with HA-Exo84 were incubated with immobilized GST-RalA G23V, subjected to GST pull-down assays, and analyzed by immunoblot. The amounts of GST fusion proteins used in the pull-down assay are shown with Ponceau S staining. Quantification of the binding of Exo84 with RalA G23V is shown. Binding was normalized to the sixth lane. The normalized ratio is represented in arbitrary units. Data are means ± SEM. *P < 0.05; n = 3 experiments; data were analyzed by Mann-Whitney U test. (C) Coimmunoprecipitates of Flag-RalA WT or G23V mutant with Exo84 or Sec5 from COS-1 cells. The interaction of endogenous Exo84 and Sec5 with RalA G23V in the presence or absence of TBK1 is shown in the square. n = 3 experiments. (D) Coimmunoprecipitates of HA-Exo84 with Flag-RalA WT, G23V, or a GDP-locked dominant-negative Ral (S28N) from COS-1 cells coexpressing TBK1 or the K38A kinase-inactive mutant. Arrow and arrowhead indicate phosphorylated Exo84 and RalA, respectively. n = 3 experiments. (E) Coimmunoprecipitates of HA-Exo84 with MYC-TBK1 WT and its kinase-inactive mutant from COS-1 cells coexpressing RalA WT, G23V, or S28N. n = 3 experiments.

  • Fig. 5 The intrinsic kinase activity of TBK1 is not regulated by insulin signaling.

    (A) TBK1 immunoprecipitates from human embryonic kidney (HEK) 293T cells expressing RalA WT, G23V, F39L, or RalB WT and F39L, or TBK1 WT and K38A were subjected to in vitro immune complex kinase assay, and phosphorylation of proteins was detected by autoradiography. As a control, immunoglobulin G (IgG) was used for immunoprecipitation. Arrows indicate phosphorylated proteins. n = 3 experiments. (B) TBK1 immunoprecipitates from 3T3-L1 adipocytes electroporated with scrambled control or TBK1 siRNA, either not treated (NT) or treated with insulin (10 nM; INS), LPS (100 ng/ml), or poly(I:C) (50 μg/ml), were subjected to in vitro immune complex kinase assay, and phosphorylation of proteins was detected by autoradiography. As a control, IgG was used for immunoprecipitation. Arrow indicates pSer396 IRF3. Quantification of relative TBK1 kinase activity is shown. TBK1 kinase activity was normalized to the second lane. The normalized ratio is represented in arbitrary units. Data are means ± SEM. *P < 0.05; n = 3 experiments; data were analyzed by Mann-Whitney U test.

  • Fig. 6 The interaction of Exo84 with RalA and other exocyst subunits requires TBK1-mediated phosphorylation of Exo84.

    (A) Schematic of Exo84 with identified TBK1 phosphorylation sites. Exo84 phosphorylation sites were identified in two independent mass spectrometry analyses with a total coverage of 60.2% of the Exo84 amino acid sequence (fig. S5). Each phosphorylation site (P-site) and residues adjacent to the phosphorylation sites are shown. (B) Cell lysates from COS-1 cells coexpressing Flag-TBK1 WT and the K38A kinase-inactive mutant with HA-Exo84 WT, S8A, and S8E were incubated with immobilized GST-RalA G23V, subjected to GST pull-down assays, and analyzed by immunoblot. The amounts of GST fusion proteins used in the pull-down assay are shown with Ponceau S staining. Quantification of the binding of Exo84 WT and its mutants with RalA G23V is shown. Binding was normalized to the second lane. The normalized ratio is represented in arbitrary units. Data are means ± SEM. *P < 0.05; n = 3 experiments; data were analyzed by Mann-Whitney U test. (C) Coimmunoprecipitates of HA-Exo84 WT, S8A, and S8E with Sec5 and Sec8 from COS-1 cells coexpressing Flag-TBK1 WT and its kinase-inactive mutant. n = 3 experiments. Arrows indicate Sec8.

  • Fig. 7 Phosphorylation of Exo84 by TBK1 is essential for insulin-stimulated GLUT4 trafficking.

    (A) Immunostaining of 3T3-L1 adipocytes stably expressing MYC-GLUT4-eGFP electroporated with HA-tagged Exo84 WT, S8A, and S8E and stimulated with 10 nM insulin for 15 min. MYC (red) in nonpermeabilized cells indicates GLUT4 translocation to the plasma membrane. HA (cyan) in permeabilized cells indicates Exo84 overexpression. GFP fluorescence in cells indicates total GLUT4 expressed. A merge of GFP, HA, and MYC is shown. Arrowhead indicates cells expressing Exo84. Scale bar, 20 μm. n = 3 experiments. (B) Percentage of cells that undergo GLUT4 translocation as shown in (A). Data are means ± SEM. *P < 0.05; n = 90 to 135 cells per group; data were analyzed by Mann-Whitney U test. (C) Immunoblot showing overexpression of HA-Exo84 WT, S8A, and S8E from (A). n = 3 experiments.

  • Fig. 8 Temporal and spatial dynamics of the TBK1/exocyst/RalA complex in insulin-stimulated GLUT4 trafficking.

    (A) Exo84 immunoprecipitates from 3T3-L1 adipocytes pretreated or not with amlexanox (50 μM) before being treated with or without insulin (10 nM). As a control, goat serum (GS) was used for immunoprecipitation. n = 3 experiments. (B) Cell lysates from 3T3-L1 adipocytes treated with or without insulin (10 nM) in the presence or absence of pretreatment with amlexanox (50 μM) were incubated with GST-RalBP1 RBD beads, subjected to GST pull-down assays, and analyzed by immunoblot. The amounts of GST fusion proteins used in the pull-down assay are shown with Ponceau S staining. n = 3 experiments. (C) Subcellular fractionation of 3T3-L1 adipocytes treated with or without insulin (10 nM) for 15 min was subjected to immunoblot. n = 3 experiments. N/M, nuclear and mitochondrial remnants; PM, plasma membrane; HDM, high-density microsomes; Cyt, cytosol. Arrows indicate specific proteins as indicated. (D) 3T3-L1 adipocytes treated with or without insulin (10 nM) for 15 min were subjected to nondetergent extraction and sucrose density gradient fractionation. All fractions were subjected to immunoblot. n = 3 experiments.

  • Fig. 9 TBK1 deficiency attenuates insulin-stimulated glucose uptake in isolated adipocytes from epididymal fat.

    (A) Adipocytes from TBK1 FL and adipocyte-specific TBK1 AKO mice were subjected to glucose uptake assays with [14C]glucose. Radioactivity counts were normalized to control for each group. Data are means ± SEM. *P < 0.05; n = 3 mice per condition. Data are analyzed by Student’s t test. (B) Immunoblots from (A) show phosphorylation of Akt. Results were quantified by ImageJ. (C) Proposed model for the role of TBK1-mediated Exo84 phosphorylation in insulin-stimulated GLUT4 trafficking. Insulin activates RalA as the RGC1/2 is persistently turned off by insulin-dependent phosphorylation mediated by Akt. After activation of RalA by insulin, the G protein binds to both Sec5 and Exo84, thus engaging and directing GLUT4 vesicles to regions on the plasma membrane where fusion machineries are enriched. In addition, direct phosphorylation of the exocyst subunit Exo84 by TBK1 disengages the effector protein from RalA and the rest of the exocyst subunits, in the process ensuring that GLUT4 trafficking is not arrested at the plasma membrane.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/471/eaah5085/DC1

    Fig. S1. TBK1 activity is required for insulin-stimulated GLUT4 trafficking.

    Fig. S2. TBK1 phosphorylates Exo84 both in vitro and in cells.

    Fig. S3. TBK1 directly interacts with Exo84 through the coiled-coil domain of TBK1.

    Fig. S4. TBK1 does not affect RalA activity.

    Fig. S5. Mass spectrometry sequence coverage of affinity-purified phosphorylated HA-Exo84.

    Fig. S6. Temporal dynamics of the TBK1/exocyst/RalA complex in 3T3-L1 adipocytes in response to insulin.

    Data files S1 and S2. Identified phosphorylation sites of Exo84 from mass spectrometry.

  • Supplementary Materials for:

    Phosphorylation of the exocyst protein Exo84 by TBK1 promotes insulin-stimulated GLUT4 trafficking

    Maeran Uhm, Merlijn Bazuine, Peng Zhao, Shian-Huey Chiang, Tingting Xiong, Sheelarani Karunanithi, Louise Chang, Alan R. Saltiel*

    *Corresponding author. Email: asaltiel{at}ucsd.edu

    This PDF file includes:

    • Fig. S1. TBK1 activity is required for insulin-stimulated GLUT4 trafficking.
    • Fig. S2. TBK1 phosphorylates Exo84 both in vitro and in cells.
    • Fig. S3. TBK1 directly interacts with Exo84 through the coiled-coil domain of TBK1.
    • Fig. S4. TBK1 does not affect RalA activity.
    • Fig. S5. Mass spectrometry sequence coverage of affinity-purified phosphorylated HA-Exo84.
    • Fig. S6. Temporal dynamics of the TBK1/exocyst/RalA complex in 3T3-L1 adipocytes in response to insulin.

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    Format: Adobe Acrobat PDF

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    Other Supplementary Material for this manuscript includes the following:

    • Data files S1 and S2 (Microsoft Excel format). Identified phosphorylation sites of Exo84 from mass spectrometry.

    Citation: M. Uhm, M. Bazuine, P. Zhao, S.-H. Chiang, T. Xiong, S. Karunanithi, L. Chang, A. R. Saltiel, Phosphorylation of the exocyst protein Exo84 by TBK1 promotes insulin-stimulated GLUT4 trafficking. Sci. Signal. 10, eaah5085 (2017).

    © 2017 American Association for the Advancement of Science

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